The chromatin structure of morphologically-similar, but increasingly-malignant erythroleukemia cells was investigated using milk micrococcal nuclease digestion of isolated nuclei. The maximum solubilization of chromatin was unique for each of the three cell types: the least malignant (our Stage II) released 61% of its chromatin DNA, the most malignant (Stage IV), 46%, and the intermediate (Stage III) released 36%. An analysis of the nucleosome oligomers liberated by digestion also demonstrated differences. After 15 minutes of digestion when release was reaching its maximum, a greater proportion of large nucleosomal oligomers (sizes > trinucleosome) was released from Stage II nuclei than from Stage III or IV nuclei. The cell types also differed in the relative amount of H1-depleted mononucleosomes released. Analysis of the size of the double-stranded DNA associated with mononucleosomal particles showed that Stage III mononucleosomes were smaller (148 bp) than Stage IV (167 bp) or Stage II (190 bp). In addition, while the DNA of mononucleosomes depleted in H1 was smaller than that in the H1-containing species, relative size differences among the different cell types were retained. These data suggested that the difference in the mononuocleosome particle size resistant to nuclease digestion was independent of histone H1. Differences in nucleosome repeat length were also noted among the cell types. These studies have demonstrated dramatic differences in chromatin structure associated with malignant potential of an otherwise morphologically identical cell type. These findings may reflect changes in the relative amounts of H2a variants which we have previously described among the different malignant cell types.
Ethidium bromide (10 mug/ml) and bromodeoxyuridine (25 mug/ml) were used to sensitize selective cell organelles to visible wavelengths of an argon ion laser (488 and 514 nanometers). Ethidium bromide was shown to be selective in sensitizing nucleoli, chromosomes, and the centriolar region of PTK2 cells to the laser microbeam. Similarly, BrDU sensitized chromosomes to the microbeam irradiation. The lesions produced on the chromosomes when either agent was used appeared as a phase paling of the irradiated segment. Nucleolar lesions also appeared as a phase paling, and the centriolar region alteration appeared either as a phase paling or a phase darkening.
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